Hepatitis E virus (HEV), species Paslahepevirus balayani, belongs to the genus Paslahepevirus of the family Hepeviridae (International Committee on the Taxonomy of Viruses). Eight genotypes have been defined, of which genotypes 1 and 2 only infect humans, whilst genotypes 3-6 infect mainly pigs and wild boars, with zoonotic spillover infections from genotypes 3 and 4 to humans [1], [2], [3], [4], [5], [6], [7] .Genotypes 7 and 8 are found in old world camelids [8,9].

The single-stranded, polyadenylated RNA genome of ∼7.2 kb length encodes three open reading frames (ORF). ORF1 encodes non-structural proteins and is of variable length due to common host genome-derived insertions or deletions within a hypervariable region (HVR) [10], [11], [12]. ORF2 encodes the capsid protein, and ORF3 the multifunctional protein VP13 [13]. Genotype 1 encodes an additional ORF4 [14].

Annually, HEV infects ∼20 million people, with ∼56,000 fatalities [15,16]. Usually, the infection is self-limiting, however, chronic infections can occur among immunocompromised, such as transplant patients, and may lead to severe disease [17], [18], [19]. On rare occasions, HEV invades the central nervous system (CNS), leading to encephalitis, meningitis, and other neurological disorders [20,21]. In patients with chronic infections, ribavirin treatment is the standard of care [22]. However, treatment failure due to resistance mutations is common [23]. Prior to treatment, sequencing and analysis of HEV diversity in patients might allow predictions regarding treatment success/failure. For such predictions, partial genome sequencing e.g. by Sanger sequencing of RT-PCR amplicons is unlikely to be sufficient because it lacks the sensitivity to detect minor viral populations carrying resistance mutations. Here, high-throughput sequencing (HTS) approaches are of benefit. However, HTS, especially from clinical sample material, is challenging, given low viral loads, high host-background, or due to the presence of viral quasispecies.

In this study, we address these challenges for HEV sequencing, by using samples with a potential new subtype, degraded samples, and tissue samples from a fatal HEV infection.